Process for concentrating protein with disease-related conformation

ABSTRACT

A method of concentrating a disease-related conformation of a protein such as the PrP Sc  in a sample is disclosed. The method comprises liquefying the sample and adding a complexing agent such as phosphotungstic acid (PTA) which complexes preferentially or exclusively with the PrP Sc . After the complex is formed the composition is centrifuged until the complex settles at the bottom. Thereafter, the supernatant is poured away. The remaining pellet may be resuspended in an aqueous solution containing a protease inhibitor for storage. The PTA stains the PrP Sc  making the resulting concentrated PrP Sc  susceptible to further analysis, making it possible to quickly and efficiently determine the presence of PrP Sc  and its concentration in a sample. The method can be used to render a sample non-infectious by removing all or substantial of the infectious form of a protein from a sample.

FIELD OF THE INVENTION

This invention relates generally to methods of treating proteins andmore specifically to methods of concentrating a particularly desiredprotein within a sample.

BACKGROUND OF THE INVENTION

Prions are infectious pathogens that cause invariably fatal priondiseases (spongiform encephalopathies) of the central nervous system inhumans and animals. Prions differ significantly from bacteria, virusesand viroids. The dominating hypothesis is that no nucleic acid isnecessary to allow for the infectivity of a prion protein to proceed.

A major step in the study of prions and the diseases they cause was thediscovery and purification of a protein designated prion protein[Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Boltonet al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983)Cell 35:57-62]. Complete prion protein-encoding genes have since beencloned, sequenced and expressed in transgenic animals. PrP^(c) isencoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell46:417-428] and when PrP^(c) is expressed it is generally found on theouter surface of neurons. Many lines of evidence indicate that priondiseases results from the transformation of the normal form of prionprotein (PrP^(c)) into the abnormal form (PrP^(Sc)). There is nodetectable difference in the amino acid sequence of the two forms.However, PrP^(Sc) when compared with PrP^(c) has a conformation withhigher β-sheet and lower α-helix content [Pan, Baldwin et al. (1993)Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) JBiol Chem 268:20276-20284]. The presence of the abnormal PrP^(Sc) formin the brains of infected humans or animals is the only disease-specificdiagnostic marker of prion diseases.

PrP^(Sc) plays a key role in both transmission and pathogenesis of priondiseases (spongiform encephalopathies) and it is a critical factor inneuronal degeneration [Prusiner (1997) The Molecular and Genetic Basisof Neurological Disease, 2nd Edition: 103-143]. The most common priondiseases in animals are scrapie of sheep and goats and bovine spongiformencephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr TopMicrobiol Immunol 172:21-38]. Four prion diseases of humans have beenidentified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Streussler-Sheinker Disease (GSS), and (4) fatal familialinsomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschleret al. (1992) N Engl J Med 326:444-449]. Initially, the presentation ofthe inherited human prion diseases posed a conundrum which has sincebeen explained by the cellular genetic origin of PrP.

Most CJD cases are sporadic, but about 10-15% are inherited as autosomaldominant disorders that are caused by mutations in the human PrP gene[Hsiao and Prusiner (1990) Neurology 40:1820-1827; Goldfarb, Petersen etal. (1992) Science 258:806-808; Kitamoto and Tateishi (1994) PhilosTrans R Soc Lond B 343:391-398]. However, the human prion diseases arealso infectious; the first recognized example being kuru which isbelieved to spread in New Guinea highlands by ritualistic cannibalism.Another example of human-to-human transmission are cases of iatrogenicCJD, caused by human growth hormone derived from cadaveric pituitariesas well as dura mater grafts [Brown, Preece et al. (1992) Lancet340:24-27]. A newly perceived threat of human infection arises in therecent cases of variant CJD with the possible transmission of prionsfrom BSE-infected cows. The seriousness of the health risk resultingfrom the lack of a direct prion assays in different body fluids, tissuesamples or human- and animal-derived pharmaceuticals is exemplifiedbelow.

More than 75 young adults who were previously treated with (HGH) humangrowth hormone derived from human pituitaries have developed CJD [Koch,Berg et al. (1985) N Engl J Med 313:731-733; Buchanan, Preece et al.(1991) Br Med J 302:824-828; Fradkin, Schonberger et al. (1991) JAMA265:880-884; Brown, Preece et al. (1992) Lancet 340:24-27]. Fortunately,recombinant HGH is now used, although the seemingly remote possibilityhas been raised that increased expression of wild-type PrP^(c)stimulated by high HGH might induce prion disease [Lasmezas, Deslys etal. (1993) Biochem Biophys Res Commun 196:1163-1169]. The conclusionthat the HGH prepared from pituitaries was contaminated with prions, issupported by the transmission of prion disease to a monkey 66 monthsafter inoculation with a suspect lot of HGH [Gibbs, Asher et al. (1993)N Engl J Med 328:358-359]. Because of the long incubation timesassociated with prion diseases it will not be possible to determine thefull extent of iatrogenic CJD in thousand of people treated with HGHworldwide for decades. Iatrogenic CJD also appears to have developed infour infertile women treated with contaminated human pituitary-derivedgonadotropin hormone [Cochius, Mack et al. (1990) Aust N Z J Med20:592-593; Cochius, Hyman et al. (1992) J Neurol Neurosurg Psychiatry55:1094-1095; Healy and Evans (1993) Br J Med 307:517-518] as well as atleast 11 patients receiving dura mater grafts [Thadani, Penar et al.(1988) J Neurosurg 69:766-769; Nisbet, MacDonaldson et al. (1989) J AmMed Assoc 261:1118; Willison, Gale et al. (1991) J Neurol NeurosurgPsychiatry 54:940; Brown, Preece et al. (1992) Lancet 340:24-27]. Thesecases of iatrogenic CJD underscore the need to screen pharmaceuticalsthat might possibly be contaminated with prions.

Recently, two physicians in France were charged with involuntarymanslaughter of a child who had been treated with growth hormonesextracted from corpses. The child developed Creutzfeldt-Jakob Disease(see New Scientist, Jul. 31, 1993, page 4). According to the PasteurInstitute, since 1989 there have been 24 reported cases of CJD in youngpeople who were treated with human growth hormone between 1983 andmid-1985. Fifteen of these children have died. It appears that hundredsof children in France have been treated with growth hormone extractedfrom dead bodies that were at risk for developing CJD (see NewScientist, Nov. 20, 1993, page 10).

Another major concern is the epidemic of BSE in Great Britain andadditional cases in some other countries of European Community[Wilesmith (1996) Methods in Molecular Medicine: Prion Diseases:155-173]. The epidemic spread in the early 80s was probably due to therecycling of prion-infected animals in the rendering process and thefeeding of cattle with prion-contaminated protein supplement. Theenormous economic cost of eradication of BSE, if ever completelypossible [Anderson, Donnelly et al. (1996) Nature 382:779-788], is nowoutweighed by the discovery of new variant CJD in young people in GreatBritain which was probably transmitted by consumption ofBSE-contaminated beef [Collinge, Beck et al. (1996) Lancet 348:56;Collinge, Sidle et al. (1996) Nature 383:685-690; Will, Ironside et al.(1996) Lancet 347:921-925]. Because of the long incubation time of CJD,it is too early to estimate the true extent of threat to the generalpopulation in Great Britain and the rest of the Europe from theavailable epidemiology. The BSE epidemic in cows, the "new variant" CJDand all the cases of iatrogenic CJD in young people underscore the needfor screening food sources and pharmaceuticals that might possibly becontaminated with prions.

The most sensitive method today to detect and measure prions is bioassayin transgenic animals overexpressing the cellular prion protein PrP^(c).The current prion titrations are performed in two steps: (1) the samplematerial is first injected into susceptible experimental animals toamplify prions and PrP^(Sc) protein to detectable levels; (2) theclinically symptomatic animals are euthanized and the disease isverified by detecting disease-specific PrP^(Sc) and pathology. Since thediscovery of protease resistance of PrP^(Sc) more than 15 years ago, thePrP^(Sc) detection is exclusively based on protease treatment of brainsamples with proteinase K; the residual C-terminal protease-resistantfragment PrP 27-30 is then detected in denatured form by polyclonal ormonoclonal antibodies recognizing prion protein on Western blots. Morerecent modifications of the same principle are semiquantitative dotblots or qualitative histoblots [Serban, Taraboulos et al. (1990)Neurology 40:110-117; Taraboulos, Jendroska et al. (1992) Proc Natl AcadSci USA 89:7620-7624].

Despite the dramatic shortening of incubation time of human prions intransgenic mice overexpressing chimeric or human PrP genes, in somecases to less than 120 days, the potential for broad and highflow-through application of such prion bioassays is still limited. Onepossibility further shortening the assay time is to increase thesensitivity of PrP^(Sc) detection. This would shorten the necessaryobservation time, increase the flow-through and as a result, make assaysless expensive and broadly applicable.

A system for detecting PrP^(Sc) by enhancing immunoreactivity afterdenaturation is provided in Serban, et al., Neurology, Vol. 40, No. 1,Ja 1990. Sufficiently sensitive and specific direct assay for infectiousPrP^(Sc) in biological samples could potentially abolish the need foranimal inoculations completely. Unfortunately, such does not appear tobe possible with current PrP^(Sc) assays--it is estimated that thecurrent sensitivity limit of proteinase-K and Western blot-basedPrP^(Sc) detection is in a range of 1 μg/ml which corresponds to 10⁴-10⁵ prion infectious units. Additionally, the specificity of thetraditional proteinase-K-based assays for PrP^(Sc) is in question inlight of recent findings of only relative or no proteinase-K resistanceof undoubtedly infectious prion preparations [Hsiao, Groth et al. (1994)Proc Natl Acad Sci USA 91:9126-9130] Telling, et al. (1996) Genes & Dev.

Human transthyretin (TTR) is a normal plasma protein composed of fouridentical, predominantly β-sheet structured units, and serves as atransporter of hormone thyroxin. Abnormal self assembly of TTR intoamyloid fibrils causes two forms of human diseases, namely senilesystemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP)[Kelly (1996) Curr Opin Strut Biol 6(1):11-7]. The cause of amyloidformation in FAP are point mutations in the TTR gene; the cause of SSAis unknown. The clinical diagnosis is established histologically bydetecting deposits of amyloid in situ in bioptic material.

To date, little is known about the mechanism of TTR conversion intoamyloid in vivo. However, several laboratories have demonstrated thatamyloid conversion may be simulated in vitro by partial denaturation ofnormal human TTR [McCutchen, Colon et al. (1993) Biochemistry32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun197(2) 415-21]. The mechanism of conformational transition involvesmonomeric conformational intermediate which polymerizes into linearβ-sheet structured amyloid fibrils [Lai, Colon et al. (1996)Biochemistry 35(20):6470-82]. The process can be mitigated by bindingwith stabilizing molecules such as thyroxin or triiodophenol [Miroy, Laiet al. (I1996) Proc Natl Acad Sci USA 93(26):15051-6].

In view of the above points, there is clearly a need for a specific,high flow-through, and cost-effective assay for testing sample materialsfor the presence of infectious form of prion protein, PrP^(Sc), which isbelieved to be the cause of prion diseases, such as BSE, CJD andscrapie. The presented invention offers a method of improvingsensitivity of a range of different assays.

SUMMARY OF THE INVENTION

A method of concentrating a disease conformation of a protein such asthe PrP^(Sc) in a sample is disclosed. The method comprises liquefyingthe sample and adding a complexing agent such as phosphotungstic acid(PTA) which complexes with the PrP^(Sc). After the complex is formed thecomposition is centrifuged until the complex settles at the bottom.Thereafter, the supernatant is poured away. The remaining pellet may beresuspended in an aqueous solution containing a protease inhibitor forstorage. The PTA stains the PrP^(Sc) making the resulting concentratedPrP^(Sc) susceptible to further analysis. The original sample has beencleansed of PrP^(Sc) to the extent that a sample originally possessinginfectivity is rendered non-infective.

An object of the invention is to provide a method for concentrating thedisease conformation of a protein which has two or more conformationalforms.

A specific object of the invention is to provide a method forconcentrating PrP^(Sc) within a sample.

Another object is to reduce or completely eliminate the infectivity of asample as regards prion diseases.

An advantage of the invention is that it can be quickly and convenientlycarried out without the use of complex procedures or devices.

Another advantage is that materials containing infective amounts ofPrP^(Sc) can be rendered non-infective.

A feature of the invention is that PrP^(Sc) forms complexes almostexclusively with phosphotungstic acid.

An important object of the method is to concentrate infectious prionspresent from crude mixtures such as brain homogenates or in variablesample materials obtained or derived from human, primate, monkey,hamster, mice, pig, bovine, sheep, deer, elk, cat, dog, and chickentissues.

Another object is to provide a simple, fast, and inexpensive method toimprove the safety of biologicals by eliminating infectious prions fromsamples potentially contaminated with prions.

Another object is to provide materials such as organic polymers,inorganic salt complexes, or metals modified by phosphotungstic acidwhich materials are useful in concentrating or eliminating prions fromsuch crude mixtures.

An important feature and advantage of the method is the rapid,cost-effective and high-through design with the capacity to process morethan 100 samples per day per skilled person.

These and other objects, advantages and features of the invention willbecome apparent to those skilled in the art upon reading thisdisclosure.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a bar graph of the prion titer (log ID₅₀ /ml) for fourdifferent samples tested.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present assays and methods are disclosed and described, it isto be understood that this invention is not limited to particularcomplexing agents, proteins, labels, assays or method as such may, ofcourse vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for the disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided are subject to change if itis found that the actual date of publication is different from thatprovided here.

DEFINITIONS

The term "complexing agent" is used herein to refer to any materialwhich binds or complexes selectively with the constrictive conformationof a protein (e.g. with PrP^(Sc)) and not with the relaxed conformationof a protein (e.g. PrP^(c)). The resulting complex has a higher densitythan either the constrictive conformation or the complexing agent alone.A preferred complexing agent for PrP^(Sc) is phosphotungstic acid (PTA),which may be added in the form of a salt, e.g. sodium phosphotungstate.The complexing agent preferably forms a complex which has a higherdensity than the density of the constricted conformation of the proteinby itself. This makes it possible to precipitate the complex out bycentrifugation. The complex formed must provide some means forseparating the complex from the remainder of the composition.

The term "protein" as used herein is intended to encompass any aminoacid sequence and include modified sequences such as glycoproteins. Theterm includes naturally occurring proteins and peptides as well as thosewhich are recombinantly or synthetically synthesized. As used inconnection with the present invention the term "protein" is specificallyintended to cover naturally occurring proteins which occur in at leasttwo different conformations, a "constricted conformation" and a "relaxedconformation," wherein both conformations have the same or substantiallythe same amino acid sequence but have different three dimensionalstructures. The two conformations of the protein may include at leastone conformation which is not related to a disease state and at leastone conformation which is related to a disease state. A specific andpreferred example of a protein as used in connection with thisdisclosure is a PrP protein which includes a relaxed conformation (thenon-disease form), referred to as PrP^(c), and the constrictedconformation (the disease related form), referred to as PrP^(Sc).

The terms "treating", "treatment" and the like are used interchangeablyhere to describe a process whereby a sample or portion thereof andspecifically proteins in the sample are physically and/or chemicallymanipulated so that proteins in the sample in a disease relatedconformation are caused to changed to a different conformation withhigher affinity for a binding partner, e.g., a higher antibody bindingaffinity. Treated proteins are also referred to as denatured proteins orproteins in a relaxed conformation which conformation increases theantibody binding affinity of the protein. Treating includes subjectingthe sample to heat, pressure and/or chemicals. In a preferredembodiment, samples containing PrP^(Sc) (which is the disease-relatedconformation comprising β-sheet structural configurations) are treatedso that the PrP^(Sc) protein assumes a different conformation (e.g.,comprising an α-helical configuration and/or a random coilconfiguration) having four times or more greater antibody bindingaffinity.

The terms "PrP protein", "PrP" and like are used interchangeably hereinand shall mean both the infectious particle form PrP^(Sc) known to causediseases (spongiform encephalopathies) in humans and animals and thenoninfectious form PrP^(c) which, under appropriate conditions isconverted to the infectious PrP^(Sc) form.

The terms "prion", "prion protein" and "PrP^(Sc) protein" and the likewe used interchangeably herein to refer to the infectious PrP^(Sc) formof a PrP protein, and is a contraction of the words "protein" and"infection." Particles are comprised largely, if not exclusively, ofPrP^(Sc) molecules encoded by a PrP gene. Prions are distinct frombacteria, viruses and viroids. Known prions infect animals to causescrapie, a transmissible, degenerative disease of the nervous system ofsheep and goats, as well as bovine spongiform encephalopathy (BSE), or"mad cow disease", and feline spongiform encephalopathy of cats. Fourprion diseases known to affect humans are (1) kuru, (2)Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-ScheinkerDisease (GSS), and (4) fatal familial insomnia (FFI). As used herein"prion" includes all forms of prions causing all or any of thesediseases or others in any animals used--and in particular in humans anddomesticated farm animals.

The term "PrP gene" is used herein to describe genetic material whichexpresses proteins including known polymorphisms and pathogenicmutations. The term "PrP gene" refers generally to any gene of anyspecies which encodes any form of a prion protein. Some commonly knownPrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci.USA 89:9097-9101 (1992) and U.S. Pat. No. 5,565,186, incorporated hereinby reference to disclose and describe such sequences. The PrP gene canbe from any animal, including the "host" and "test" animals describedherein and any and all polymorphisms and mutations thereof, it beingrecognized that the terms include other such PrP genes that are yet tobe discovered. The protein expressed by such a gene can assume either aPrP^(c) (non-disease) or PrP^(Sc) (disease) form.

The terms "standardized prion preparation", "prion preparation","preparation" and the like are used interchangeably herein to describe acomposition obtained from the brain tissue of mammals which exhibitssigns of prion disease: the mammal either (1) include a transgene asdescribed herein; (2) have and ablated endogenous prion protein gene;(3) have a high number of prion protein gene from a genetically diversespecies; or (4) are hybrids with an ablated endogenous prion proteingene and a prion protein gene from a genetically diverse species.Different combinations of 1-4 are possible, e.g., 1 and 2. The mammalsfrom which standardized prion preparations are obtained exhibit clinicalsigns of CNS dysfunction as a result of inoculation with prions and/ordue to developing the disease of their genetically modified make up,e.g., high copy number of prion protein genes.

The term "artificial PrP gene" is used to encompass the term "chimericPrP gene" as well as other recombinantly constructed genes which, whenincluded in the genome of a host animal (e.g. a mouse, will render themammal susceptible to infection from prions which naturally only infecta genetically diverse test animal, e.g. human, bovine or ovine. Ingeneral, an artificial gene will include the codon sequence of the PrPgene of the mammal being genetically altered with one or more (but notall, and generally less than 40) codons of the natural sequence beingreplaced with a different codon--preferably a corresponding codon of agenetically diverse mammal (such as a human). The genetically alteredmammal being used to assay samples for prions only affect thegenetically diverse mammal. Examples of artificial genes are mouse PrPgenes encoding the sequence for human, cows and sheep and replacingmouse codons at the same relative positions, with the provision that notall the mouse codons are replaced with differing human, cow or sheepcodons. Artificial PrP genes can include not only codons of geneticallydiverse animals but may include codons and codon sequences notassociated with any native PrP gene but which, when inserted into ananimal render the animal susceptible to infection with prions whichwould normally only infect an genetically diverse animal.

The terms "chimeric gene", "chimeric PrP gene", "chimeric prion proteingene" and the like are used interchangeably herein to refer to anartificially constructed gene containing the codons of a host animalsuch as a mouse with one or more of the codons replaced withcorresponding codons from a genetically diverse test animal such as ahuman, cow or sheep. In one specific example, the chimeric gene iscomprised of the starting and terminating sequence (e.g.,--and C-terminal codons) of PrP gene of a mammal of host species (e.g. a mouse)and also containing a nucleotide sequence of a corresponding portion ofa PrP gene of a test mammal of a second species (e.g. a human). When achimeric gene is inserted into the genome of the host species, it willrender the mammal susceptible to infection with prions which normallyinfect only mammals of the second species. The preferred chimeric genedisclosed herein is MHu2M which contains the starting and terminatingsequence of a mouse PrP gene and a non-terminal sequence region which isreplaced with a corresponding human sequence differing from a mouse PrPgene at nine residues.

The term "genetic material related to prions" is intended to cover anygenetic material which affects the ability of an animal to becomeinfected with prions. Thus the term encompasses any "PrP gene","artificial PrP gene", "chimeric PrP gene" or "ablated PrP gene" whichterms are defined herein as well as modification of such which effectthe ability of an animal to become infected with prions. Standardizedprion preparations are produced using animals which all havesubstantially the same genetic material related to prions so that all ofthe animals will become infected with the same type of prions and willexhibit signs of infection at approximately the same time.

The term "host animal" and "host mammal" are used to describe animalswhich will have their genome genetically and artificially manipulated soas to include genetic material which is not naturally present within theanimal. For example, host animals include mice, hamsters and rats whichhave their PrP gene ablated, i.e., rendered inoperative. The host isinoculated with prion proteins to generate antibodies. The cellsproducing the antibodies are a source of genetic material for making aphage library. Other host animals may have a natural (PrP) gene, or onewhich is altered by the insertion of an artificial gene or by theinsertion of a native PrP gene of a genetically diverse test animal.

The term "test animal" and "test mammal" are used to describe the animalwhich is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith which the test animal would generally be susceptible to infection.For example, the test animal may be a human, cow, sheep, pig, horse,cat, dog or chicken, and one may wish to determine whether a particularsample includes prions which would normally only infect the test animal.

The terms "genetically diverse animal" and "genetically diverse mammal"are used to describe an animal which includes a native PrP codonsequence of the host animal which differs from the genetically diversetest animal by 17 or more codons, preferably 2 or more codons, and mostpreferably 28-40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a human, cow or sheep, but is notgenetically diverse with respect to the PrP gene of a hamster.

The term "ablated PrP protein gene", "disrupted PrP gene", and the likeare used interchangeably herein to mean an endogenous PrP gene which hasbeen altered (e.g., add and/or remove nucleotides) in a manner so as torender the gene inoperative. Examples of non-functional PrP genes andmethods of making such are disclosed in Bueler, H., et al "Normaldevelopment of mice lacing the neuronal cell-surface PrP protein" Nature356:577-582 (1992) and Weissmann (WO93/10227). The methodology forablating a gene is taught in Capecchi, Cell 51:503-512 (1987), all ofwhich are incorporated herein by reference. Preferably both alleles ofthe genes are disrupted as represented by PrP^(0/0) or Prnp^(0/0).

The terms "hybrid animal", "transgenic hybrid animal" and the like areused interchangeably herein to mean an animal obtained by thecross-breeding of a first animal having an ablated endogenous prionprotein gene with a second animal which includes either (1) a chimericgene or artificial PrP gene or (2) a PrP gene from a genetically diverseanimal. For example a hybrid mouse is obtained by cross-breeding a mousecontaining an ablated mouse gene with a mouse containing (1) human PrPgenes (which may be present in high copy numbers) or (2) chimeric genes.The term hybrid includes any offspring of a hybrid including inbredoffspring of two hybrids provided the resulting offspring is susceptibleto infection with prions with normal infect only a genetically diversespecies. A hybrid animal can be inoculated with prions and serve as asource of cells for the creation of hybridomas to make monoclonalantibodies of the invention.

The terms "susceptible to infection" and "susceptible to infection byprions" and the like are used interchangeably herein to describe atransgenic or hybrid test animal which develops a disease if inoculatedwith prions which would normally only infect a genetically diverse testanimal. The terms are used to describe a transgenic or hybrid animalsuch as a transgenic mouse Tg(MHu2M) which, without the chimeric PrPgene, would not come infected with a human prion but with the chimericgene is susceptible to infection with human prions.

The term "non-infectious" means that the treated material does not causeinfection. More specifically, a material is infectious if it containssufficient amounts of PrP^(Sc) such that when it is used to innoculatean animal that animal will become ill with a prion disease and would nothave become ill but for the innoculation. If that material is treatedper the present invention, sufficient PrP^(Sc) can be removed such thatthe material would not cause a prion disease if used to innoculate ananimal and as such has been rendered "non-infectious."

The term "antibody" stands for an immunoglobulin protein which iscapable of binding an antigen. Antibody as used herein is meant toinclude the entire antibody as well as any antibody fragments (e.g.F(ab)', Fab, Fv) capable of binding the epitope, antigen or antigenicfragment of interest. Preferred antibodies for assays of the inventionare immunoreactive or immunospecific for and therefore specifically andselectively bind to a protein of interest e.g., an A4β amyloid proteinor a PrP protein. Antibodies which are immunoreactive and immunospecificfor both the native non-disease form and the treated disease form butnot for the untreated disease form, (e.g., for both native PrP^(c) andtreated PrP^(Sc) but not native PrP^(Sc)) are preferred. Antibodies forPrP are preferably immunospecific--e.g., not substantiallycross-reactive with related materials. Some specific antibodies whichcan be used in connection with the invention are disclosed in publishedPCT application WO 97/10505 which is incorporated herein by reference todisclose and describe antibodies. This published PCT applicationcorresponds to U.S. Ser. No. 08/713,939 also incorporated herein byreference. Antibodies disclosed in the PCT application which selectivelybind PrP^(Sc) should not be used in the present invention. The term"antibody" encompasses all types of antibodies, e.g. polyclonal,monoclonal, and those produced by the phage display methodology.Particularly preferred antibodies of the invention are antibodies whichhave a relatively high degree of affinity for both native PrP^(c) andtreated PrP^(Sc) but a relatively low degree of or substantially nobinding affinity for PrP^(Sc). More specifically, antibodies of theinvention preferably have four times or more, more preferably fifteentimes or more, and still more preferably 30 times or more bindingaffinity for both native PrP^(c) and denatured PrP^(Sc) as compared withthe binding affinity for native PrP^(Sc).

"Purified antibody" refers to that which is sufficiently free of otherproteins, carbohydrates, and lipids with which it is naturallyassociated. Such an antibody "preferentially binds" to a treated ordenatured disease conformation of a protein such as the β-sheetconformation of A4β or PrP^(Sc) protein (or an antigenic fragmentthereof), and does not substantially recognize or bind to otherantigenically unrelated molecules. A purified antibody of the inventionis preferably immunoreactive with and immunospecific for a specificspecies and more preferably immunospecific for native PrP^(c) and fortreated or denatured forms of PrP^(c) and PrP^(Sc) but not for native oruntreated PrP^(Sc).

"Antigenic fragment" of a protein (e.g., a PrP protein) is meant aportion of such a protein which is capable of binding an antibody.

By "binds specifically" is meant high avidity and/or high affinitybinding of an antibody to a specific polypeptide e.g., epitope of aprotein, e.g., a PrP^(c) or A4β protein. Antibody binding to its epitopeon this specific polypeptide is preferably stronger than binding of thesame antibody to any other epitope, particularly those which may bepresent in molecules in association with, or in the same sample, as thespecific polypeptide of interest e.g., binds more strongly to epitopefragments of a protein such as PrP^(Sc) so that by adjusting bindingconditions the antibody binds almost exclusively to an epitope site orfragments of a desired protein such as an epitope fragment exposed bytreatment of PrP^(Sc) and not exposed on native untreated PrP^(Sc).

By "detectably labeled antibody", "detectably labeled anti-PrP" or"detectably labeled anti-PrP fragment" is meant an antibody (or antibodyfragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels known in the art, but normally are radioisotopes,fluorophores, paramagnetic labels, enzymes (e.g., horseradishperoxidase), or other moieties or compounds which either emit adetectable signal (e.g., radioactivity, fluorescence, color) or emit adetectable signal after exposure of the label to its substrate. Variousdetectable label/substrate pairs (e.g., horseradishperoxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin),methods for labeling antibodies, and methods for using labeledantibodies are well known in the art (see, for example, Harlow and Lane,eds. (Antibodies: A Laboratory Manual (1988) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Europium is a particularlypreferred label.

Abbreviations used herein include:

CNS for central nervous system;

BSE for bovine spongiform encephalopathy;

CJD for Creutzfeldt-Jacob Disease;

FFI for fatal familial insomnia;

GSS for Gerstamnn-Strassler-Scheinker Disease;

Hu for human;

HuPrP for human prion protein;

Mo for mouse;

MoPrP for mouse prion protein;

SHa for a Syrian hamster;

SHaPrP for a Syrian hamster prion protein;

Tg for transgenic;

Tg(SHaPrP) for a transgenic mouse containing the

PrP gene of a Syrian hamster;

Tg(HuPrP) for transgenic mice containing the complete human PrP gene;

Tg(ShPrP) for transgenic mice containing the complete sheep PrP gene;

Tg(BoPrP) for transgenic mice containing the complete cow PrP gene;

PrP^(Sc) for the scrapie isoform of the prion protein;

PrP^(c) for the cellular contained common, normal isoform of the prionprotein;

MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;

MHu2M for a chimeric mouse/human PrP gene wherein a region of the mousePrP gene is replaced by a corresponding human sequence which differsfrom mouse PrP at 9 codons;

Tg(MHu2M) mice are transgenic mice of the invention which include thechimeric MHu2M gene;

MHu2MPrP^(Sc) for the scrapie isoform of the chimeric human/mouse PrPgene;

PrP^(CJD) for the CJD isoform of a PrP protein;

Prnp^(0/0) for ablation of both alleles of an endogenous prion proteingene, e.g., the MoPrP gene;

Tg(SHaPrP^(+/0))81/Prnp^(0/0) for a particular line (81) of transgenicmice expressing SHaPrP, +/0 indicates heterozygous;

Tg(HuPrP)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mousewith a human prion protein gene (HuPrP with a mouse with both alleles ofthe endogenous prion protein gene disrupted;

Tg(MHu2M)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mousewith a chimeric prion protein gene (MHu2M) with a mouse with bothalleles of the endogenous prion protein gene disrupted;

TTR for transthyretin;

FVB for a standard inbred strain of mice often used in the production oftransgenic mice since eggs of FVB mice are relatively large and toleratemicroinjection of exogenous DNA relatively well;

[PrP.sub.β ]--concentration of prion protein in β-sheet conformation;

[βA4.sub.β ]--concentration of βA4 in β-sheet conformation;

[DRC]--concentration of a disease related conformation of a protein.

GENERAL ASPECTS OF THE INVENTION

Some proteins such as the protein expressed by the PrP gene have morethan one conformational shape. For example a PrP protein may assume itscellular form, i.e. PrP^(c) form or its scrapies form, i.e. PrP^(Sc)form. One form of the protein is harmless (e.g. PrP^(c)) whereas anotherform of the protein is pathogenic (e.g. PrP^(Sc)). When the constricted,pathogenic form of the protein such as PrP^(Sc) is present in an animalin very small amounts the animal is not showing symptoms of disease.However, the animal will develop a disease related to the pathogenicform of the protein--e.g. develop a prion disease. To avoid transmissionof disease it is important to determine if a sample derived from ananimal is infected with the disease form of a protein, e.g. has PrP^(Sc)in its brain tissue. The present invention is useful with respect to (1)concentrating the pathogenic form of a protein that is present in asample, (2) eliminating the pathogenic form of the protein from thesample and/or (3) reducing the concentration of the pathogenic form ofthe protein in a material to a level such that the material is rendered"non-infectious."

The presence of a pathogenic form of a protein (e.g. PrP^(Sc)) can bedetermined in a number of ways. For example a sample to be tested can beused to innoculate transgenic mice which have been geneticallyengineered to be susceptible to the pathogenic protein being testedfor--see U.S. Pat. No. 5,565,186. Alternatively, the sample can betreated to expose epitopes and tested against antibodies which bind tothe treated protein--see U.S. patent application Ser. No. 08/804,536filed Feb. 21, 1997 now U.S. Pat. No. 5,891,641 issued Apr. 6, 1999 andthe monoclonal antibody 263K 3F4, produced by cell line ATCC HB9222deposited Oct. 8, 1996 and U.S. Pat. No. 4,806,627 issued Feb. 21, 1986.

Regardless of the type of method used to assay for the presence of apathogenic form of a protein (e.g. PrP^(Sc)) the assay results can beimproved if the concentration of the pathogenic protein in the samplebeing tested can be increased. In particular, assay results can beimproved if the concentration of the pathogenic form of the protein inthe sample can be increased relative to the concentration of thenon-pathogenic form of the protein in the sample. This is particularlyimportant because the concentration of the non-pathogenic form of theprotein is generally substantially greater (10 fold or more) than theconcentration of the pathogenic form. The present invention makes itpossible to precipitate out and thereby concentrate the pathogenic formof a protein such as PrP^(Sc). Thus, the concentrated protein can beassayed thereby enhancing the sensitivity of any assay for the protein.Further, the invention can be used to reduce the concentration of apathogenic form of a protein in a material to a level such that amaterial which was infectious is rendered non-infectious.

PROCEDURES IN GENERAL

Any type of sample can be processed using the present invention in orderto concentrate the constricted, pathogenic form of a protein. However,proteins having a constrictive and a relaxed conformation are generallyfound in brain tissue. Thus, the invention is described using braintissue as the starting material from which the pathogenic protein isconcentrated. Although the invention could be applied to concentrating aconstricted form of any protein having a constricted and relaxed form,the invention is described specifically with respect to concentratingthe pathogenic form of a PrP protein, i.e. concentrating PrP^(Sc) whichis present in brain tissue along with PrP^(c).

A sample to be treated should be in a liquid flowable form at roomtemperature (15° C. to 30° C.). Brain tissue is homogenized and dilutedwith a saline solution such as a phosphate buffered saline solution. Thesolution should have a pH of about 6.4 to 8.4, preferably 7.4, notcontain magnesium or calcium and preferably comprise about 4% (w/v) ofsodium dodecylsarcosinate (Sarcosyl). The solution is added to the brainhomogenate in a ratio of 1:5 to 5:1 and preferably in a ratio of about1:1 and mixed at room temperature.

The next step is the most important in the process of the invention. Acomplexing agent is added to the sample which agent forms a complex withor somehow binds preferentially with or exclusively to any constricted(generally a pathogenic form) of the protein present in the sample.Phosphotungstic acid may be and preferably is added in the form of asalt--e.g. a stock solution containing 4% sodium phosphotungstate(NaPTA) and 170 mM MgCl₂, pH 7.4 is added to obtain a finalconcentration of PTA of about 0.2 to 0.3%. However, the PTA may be addedso as to obtain lower or higher concentrations of PTA it beingunderstood that higher concentrations can be used but are not generallyneeded to obtain the desired effect of forming complexes with anyPrP^(Sc) present. After the PTA is added the sample is subjected to asufficient amount of mixing over a period of time sufficient to allowsubstantially all the PrP^(Sc) in the sample to complex with the PTA.For example, the sample could be incubated at about 30° C. to 45° C.(preferably 37° C.) over a period of from about 1 to 16 hours on arocking platform. The PTA (which is the complexing agent) forms acomplex with the PrP^(Sc) (which is the constricted conformation of theprotein). The PTA/PrP^(Sc) complex formed will have a higher densitythan PrP^(Sc) alone. The complexing agent and protein may form any typeof complex with equal or unequal numbers of each of the molecules. Whatis important is that complex formed can be separated away from the restof the composition by some means.

Next small portions of the sample (e.g. 1 ml portions) are placed incontainers such as Eppendorf tubes and centrifuged--e.g. at about 14,000g using a table top centrifuge for about 30 minutes. It will beunderstood by those skilled in the art that lower and higher speeds canbe used over shorter or longer times to obtain the desired effect ofsettling out the heavy complexes of PrP^(Sc) /PTA formed.

Certain enzymes are capable of degrading protein in one form but notanother. For example, Proteinase K degrades PrP^(c) but not PrP^(Sc).The Proteinase K is added to the sample (before or after centrifuging)in that a low concentration of Proteinase K generally increases theefficiency of precipitation of PrP^(Sc) /PTA and thereby increasing thedensity differential between PrP^(c) (degraded) and PrP^(Sc) (complexedwith PTA).

After centrifuging, the supernatant is decanted away leaving aprecipitated pellet. The pellet is resuspended in water preferablycontaining protease inhibitors, e.g. PMSF 0.5 mM; Aprotinin andLeupeptin, 2 μg/ml each. The suspension is centrifuged and the contentof the resulting pellet is reduced 100-fold. Typically, if the samplewas taken from an animal that died from a prion disease the resultingpellet will contain about 40 to 60% PrP^(Sc) or PrP 27-30 which is acore component of PrP^(Sc) not digested by Proteinase K.

The process of the invention produces a suspension wherein the PrP^(Sc)or other pathogenic protein remaining is stained with the PTA used. Thisis desirable in that further analysis by a number of methods requiresstaining. Thus, the product obtained can be directly subjected to SDSPAGE, Western blots, dot blots or a differential conformational assay asdescribed in U.S. patent application Ser. No. 08/804,536 filed Feb. 21,1997.

The method of the invention can be used for (1) concentrating anyPrP^(Sc) in a sample for further analysis and/or (2) removing PrP^(Sc)from a material to render the material non-infectious. If rendering thematerial non-infectious is all that is required, the method of theinvention can be simplified. The material is liquified and brought intocontact with the appropriate complexing agent. After complexes have beenallowed to form centrifugation is used to precipitate out the complexesand thereby render the remaining material non-infectious. Furtherprocessing of the precipitated material is not needed but may be carriedout to obtain desired information. Such processing is described below.

FURTHER ANALYSIS

After concentrating the constricted conformation of the protein with thecomplexing agent and centrifuging as described above, the sample can andpreferably is subjected to further analysis. One such analysis protocolinvolves contacting a first portion of the sample with a bindingpartner, such as an antibody which binds PrP^(c) and treated PrP^(Sc),said binding partner having a higher affinity for the first conformation(e.g. PrP^(c)) than the second constricted conformation (e.g. PrP^(Sc)),and determining a first concentration. A suitable antibody is 3F4disclosed in U.S. Pat. No. 4,806,627 issued Feb. 21, 1986. A secondportion of the sample is treated to increase binding affinity of thesecond constructed conformation to the binding partner, for exampletreating to expose PrP^(Sc) epitopes. The treatment can involve heat,pressure and/or chemical denaturation of the constricted pathogenicprotein (e.g., PrP^(Sc)) sufficient to convert 2% or more of theconstricted form to a form which binds the binding partner. The treatedsecond portion of the sample is contacted with the binding partner todetermine a second concentration, i.e., the concentration of particleswhich now bind the binding partner. An increase should be observed ifthe original sample included protein in a constricted conformation andthat protein was converted to a different conformation due to thetreatment. After the second concentration is determined, it is adjustedto compensate for increased affinity of the protein in the firstconformation for the binding partner resulting from the treating.Finally, the first concentration is compared with the adjustedconcentration to determine the presence of protein in the secondconformation.

The first concentration and the second concentration are preferablydetermined using time-resolved, dissociation-enhanced fluorescence.Preferably, the second concentration is higher than the firstconcentration in the sample, with the second concentration being 1×10³particles/ml or less. The protein in the sample is preferably selectedfrom the group consisting of βA4 protein, PrP protein, andtransthyretin. Details regarding methods of carrying out the furtheranalysis described above are contained within U.S. patent applicationSer. No. 08/804,536 filed Feb. 21, 1997 now U.S. Pat. No. 5,891,641issued Apr. 6, 1994 and U.S. application attorney docket number06510/081001 filed Feb. 20, 1998 entitled "Assay for Disease RelatedConformation of a Protein"--both of which are incorporated by referenceto disclose and describe such methods.

DISEASES ASSOCIATED WITH INSOLUBLE PROTEINS

Much of the disclosure and the specific examples provided herein relateto the use of the invention in connection with concentrating PrP^(Sc) inthe sample. However, as indicated above, the invention can be applied todetermining the presence of any protein which assumes two or moredifferent shapes, one of which is constricted (generally associated withthe disease) and one which is relaxed (generally not a diseaseconformation). The following is a non-limiting list of diseases withassociated proteins which assume two or more different conformation--aconstricted and a relaxed conformation.

    ______________________________________                                        Disease            Insoluble Proteins                                         ______________________________________                                        Alzheimer's Disease                                                                              APP, Aβ peptide,                                         α1-antichymotrypsin,                                                    tan, non-Aβ component                                                   Prion diseases, PrP.sup.Sc                                                    Creutzfeld Jakob                                                              disease, scrapie and                                                          bovine spongeform                                                             encephalopathy                                                                ALS SOD and neurofilament                                                     Pick's disease Pick body                                                      Parkinson's disease Lewy body                                                 Diabetes Type 1 Amylin                                                        Multiple myeloma-- IgGL-chain                                                 plasma cell dyscrasias                                                        Familial amyloidotic Transthyretin                                            polyneuropathy                                                                Medullary carcinoma Procalcitonin                                             of thyroid                                                                    Chronic renal failure β.sub.2 --microglobulin                            Congestive heart failure Atrial natriuretic factor                            Senile cardiac and Transthyretin                                              systemic amyloidosis                                                          Chronic inflammation Serum amyloid A                                          Atherosclerosis ApoA1                                                         Familial amyloidosis Gelsolin                                               ______________________________________                                    

It should be noted that the insoluble proteins listed above each includea number of variants or mutations which are intended to be encompassedby the present invention. Known pathogenic mutations and polymorphismsin the PrP gene related to prion diseases are given below and thesequences of human, sheep and bovine are given in U.S. Pat. No.5,565,186, issued Oct. 15, 1996.

    ______________________________________                                        MUTATION TABLE                                                                  Pathogenic human                                                                            Human      Sheep    Bovine                                      mutations Polymorphisms Polymorphisms Polymorphisms                         ______________________________________                                        2 octarepeat insert                                                                       Codon 129  Codon 171  5 or 6 octarepeats                             Met/Val Arg/Glu                                                              4 octarepeat insert Codon 219 Codon 136                                        Glu/Lys Ala/Val                                                              5 octarepeat insert                                                           6 octarepeat insert                                                           7 octarepeat insert                                                           8 octarepeat insert                                                           9 octarepeat insert                                                           Codon 102 Pro-Leu                                                             Codon 105 Pro-Leu                                                             Codon 117 Ala-Val                                                             Codon 145 Stop                                                                Codon 178 Asp-Asn                                                             Codon 180 Val-Ile                                                             Codon 198 Phe-Ser                                                             Codon 200 Glu-Lys                                                             Codon 210 Val-Ile                                                             Codon 217 Asn-Arg                                                             Codon 232 Met-Ala                                                           ______________________________________                                    

It should also be noted that while such proteins have two different3-dimensional conformations, they have the same amino acid sequence. Oneconformation is associated with disease characteristics and is generallyinsoluble (e.g., PrP^(Sc)) whereas the other conformation is notassociated with disease characteristics (e.g., PrP^(c)) and is soluble.

SPECIFICS OF A PrP PROTEIN

The major component of purified infectious prions (PrP^(Sc))is the corewhich is designated PrP 27-30. It is this proteinase K resistant core ofthe larger native protein PrP^(Sc) which signifies the disease causingform and distinguishes this form from the ubiquitous cellular proteinPrP^(c). PrP^(Sc) is found only in scrapie infected cells whereasPrP^(c) is present in both infected and uninfected cells implicatingPrP^(Sc) as the major, if not sole, component of infectious prionparticles.

Since both PrP^(c) and PrP^(Sc) are encoded by the same single copygene, great effort has been directed toward unraveling the mechanism bywhich PrP^(Sc) is derived from PrP^(c). Central to this goal has beenthe characterization of physical and chemical differences between thesetwo molecules. Properties distinguishing PrP^(Sc) from PrP^(c) includelow solubility [Meyer, McKinley et al. (1986) Proc Natl Acad Sci USA83:2310-2314], poor antigenicity [Kascsak, Rubenstein et al. (1987) JVirol 61:3688-3693; Serban, Taraboulos et al. (1990) Neurology40:110-117], protease resistance [Oesch, Westaway et al. (1985) Cell40:735-746], and polymerization of PrP 27-30 into rod shaped aggregateswhich are very similar, on the ultrastructural and histochemical levels,to the PrP amyloid plagues seen in scrapie diseased brains [Prusiner,McKinley et al. (1983) Cell 35:349-358]. To date, attempts to identifyany post-transitional chemical modifications in PrP^(c) that lead to itsconversion to PrP^(Sc) have proven fruitless [Stahl, Baldwin et al.(1993) Biochemistry 32: 1991-2002]. Consequently, it has been proposedthat PrP^(c) and PrP^(Sc) are in fact conformational isomers of the samemolecule.

Conformational description of PrP using conventional techniques has beenhindered by problems of solubility and the difficulty in producingsufficient quantities of pure protein. However, PrP^(c) and PrP^(Sc) areconformationally distinct. Theoretical calculations based upon the aminoacid sequences of PrP proteins from several species have predicted fourputative helical motifs in the molecule. Experimental spectroscopic datawould indicate that in PrP^(c) these regions adopt α-helicalarrangements, with virtually no β-sheet [Pan, Baldwin et al. (1993) ProcNatl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1995) ResearchAdvances in Alzheimer's Disease and Related Disorders: 775-781]. Indramatic contrast, in the same study it was found that PrP^(Sc) and PrP27-30 posses significant β-sheet content, which is typical of amyloidproteins [Pan, Baldwin et al., supra; Safar, Roller et al. (1993) J BiolChem 268:20276-20284]. Moreover, studies with extended syntheticpeptides, corresponding to PrP amino acid residues 90-145, havedemonstrated that these truncated molecules may be converted to eitherα-helical or β-sheet structures by altering their solution conditions.The transition of PrP^(c) to PrP^(Sc) requires the adoption of β-sheetstructure by regions that were previously α-helical. It is believed thatthe β-sheet structural configuration does not provide exposed epitopeswhich bind well to antibodies, whereas an α-helical structuredconfiguration does provide exposed epitopes which have a higher affinityfor antibodies. Because PrP^(Sc) does not have significant exposedepitopes it is very difficult to generate antibodies which bind toPrP^(Sc) making it difficult to determine if PrP^(Sc) is present in asample or to remove PrP^(Sc) from a sample. The present inventionaddresses these difficulties.

ANTIBODIES

The complexing agent of the invention may be an antibody which antibodymay be bound to another component (e.g. a high density metal). Thatantibody may bind to PrP^(Sc), e.g. the antibody disclosed in U.S.patent application Ser. No. 08/713,939, filed Sep. 13, 1996 now U.S.Pat. No. 5,846,533 issued Dec. 8, 1998. However, in order to removePrP^(c) present in the sample, an antibody which binds selectively orexclusively to PrP^(c) may be used. Such an antibody is disclosed inU.S. Pat. No. 4,806,627, issued Feb. 21, 1989, disclosing monoclonalantibody 263K 3F4, produced by cell line ATCC HB9222 deposited on Oct.8, 1986, which is incorporated herein by reference. The cell lineproducing the antibody can be obtained from the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852.

In general, scrapie infection fails to produce an immune response, withhost organisms being tolerant to PrP^(Sc) from the same species.Antibodies which bind to either PrP^(c) or PrP^(Sc) are disclosed inWO97/10505, published Mar. 20, 1997. Any antibody binding to PrP^(c) andnot to PrP^(Sc) can be used, and those skilled in the art can generatesuch using known procedures, e.g., see methods of producing phagedisplay antibody libraries in U.S. Pat. No. 5,223,409. Polyclonalanti-PrP antibodies have though been raised in rabbits followingimmunization with large amounts of formic acid or SDS-denatured SHaPrP27-30 [Bendheim, Barry et al. (1984) Nature 310:418-421; Bode, Pocchiariet al. (1985) J Gen Virol 66:2471-2478; Safar, Ceroni et al. (1990)Neurology 40:513-517]. Similarly, a handful of anti-PrP monoclonalantibodies against PrP 27-30 have been produced in mice [Barry andPrusiner (1986) J Infect Dis 154:518-521; Kascsak, Rubenstein et al.(1987) J Virol 61:3688-3693]. These antibodies were generated againstformic acid- or SDS-denatured PrP 27-30 and are able to recognize nativePrP^(c) and treated or denatured PrP^(Sc) from both SHa and humansequally well, but do not bind to MoPrP. Not surprisingly, the epitopesof these antibodies were mapped to regions of the sequence containingamino acid differences between SHa- and MoPrP [Rogers, Yehiely et al.(1993) Proc Natl Acad Sci USA 90:3182-3186].

It is not entirely clear why many antibodies of the type described inthe above cited publications will bind to PrP^(c) and treated ordenatured PrP^(Sc) but not to native PrP^(Sc). Without being bound toany particular theory it is believed that such may take place becauseepitopes which are exposed when the protein is in the PrP^(c)conformation are unexposed or partially hidden in the PrP^(Sc)configuration--where the protein is relatively insoluble and morecompactly folded together.

For purposes of the invention an indication that no binding occurs meansthat the equilibrium or affinity constant K_(a) is 10⁶ 1/mole or less.Further, binding will be recognized as existing when the K_(a) is at 10⁷1/mole or greater, preferably 10⁸ 1/mole or greater. The bindingaffinity of 10⁷ 1/mole or more may be due to (1) a single monoclonalantibody (i.e., large numbers of one kind of antibodies) or (2) aplurality of different monoclonal antibodies (e.g., large numbers ofeach of five different monoclonal antibodies) or (3) large numbers ofpolyclonal antibodies. It is also possible to use combinationsof(1)-(3). Selected preferred antibodies will bind at least 4-fold moreavidly to the treated or denatured PrP^(Sc) forms of the protein whencompared with their binding to the native conformation of PrP^(Sc). Thefour fold differential in binding affinity may be accomplished by usingseveral different antibodies as per (1)-(3) above and as such some ofthe antibodies in a mixture could have less than a four fold difference.

A variety of different methods may be used with one or more differentantibodies. Those skill in the art will recognize that antibodies may belabeled with known labels and used with currently available robotics,sandwich assays, electronic detectors, flow cytometry, and the like.Further, the antibodies may be bound to denser components directly orvia other intermediates such as anti-antibodies.

ANTIBODY/ANTIGEN BINDING FORCES

The forces which hold an antigen and antibody together are in essence nodifferent from non-specific interactions which occur between any twounrelated proteins, i.e., other macromolecules such as human serumalbumin and human transferrin. These intermolecular forces may beclassified into four general areas which are (1) electrostatic; (2)hydrogen bonding; (3) hydrophobic; and (4) Van der Waals. Electrostaticforces are due to the attraction between oppositely charged ionic groupson two protein side-chains. The force of attraction (F) is inverselyproportional to the square of the distance (d) between the charges.Hydrogen bonding forces are provided by the formation of reversiblenon-covalent hydrogen bridges between highly electronegative elements(F, O, N, Cl), such as in the hydrophilic groups --OH, --NH₂, and--COOH. These forces are largely dependent upon close positioning of twomolecules carrying these groups. Hydrophobic forces operate in the sameway that oil droplets in water merge to form a single large drop.Accordingly, non-polar, hydrophobic groups such as the side-chains onvaline, leucine and phenylalanine tend to associate in an aqueousenvironment. Lastly, Van der Waals are forces created between moleculeswhich depend on interaction between the external electron clouds. Allthe above interactions depend on complementarity between conformation ofthe antigen and antibody recognition site.

Further information regarding each of the different types of forces canbe obtained from "Essential Immunology edited by I. M. Roitti (6thEdition) Blackwell Scientific Publications, 1988. With respect to thepresent invention, useful antibodies exhibit some or all of theseforces. It is by obtaining an accumulation of these forces in largeramounts that it is possible to obtain an antibody which has a highdegree of affinity or binding strength to the PrP protein and inparticular an antibody which has a high degree of binding strength tothe configuration of PrP^(c) and/or the random coil configurationobtained by treating any β-sheet PrP protein in situ.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees centigrade, and pressure is at or near atmospheric.

EXAMPLE 1 Purification of Hamster PrP^(c) from Normal and PrP^(Sc) FromScrapie Infected Hamster Brains

The PrP^(c) protein can be purified as described in Pan, Stahl et al.(1992) Protein Sci 1:1343-1352; Pan, Baldwin et al. (1993) Proc NatlAcad Sci USA 90:10962-10966. Protein content can be determined by aminoacid analysis. The purity of PrP^(c) protein, can be determined on SDSPAGE followed by silver staining and Western.

Standard Syrian hamster PrP^(Sc) can be purified from a standard pool ofscrapie strain Sc237 infected hamster brains as described in Turk,Teplow et al. (1988) Eur J Biochem 176:21-30. The infectivity of thisstandard, as determined by an incubation time assay on Syrian hamstersafter intracerebral inoculation, should be 10⁷.3 ID₅₀ /ml and specificinfectivity 10⁸.2 ID₅₀ /mg of PrP^(Sc) protein. However, the specificinfectivity may vary from lot to lot ±10⁰.5 ID₅₀ /mg. The proteincontent can be determined by BCA assay using Bovine serum albumin as astandard. The preparation can be considered homogeneous with one majorband on SDS PAGE after silver staining and Western Blots. The PrPproteins of the brain of other animals can be obtained in the samemanner.

EXAMPLE 2 Isolation of PrP^(Sc) from Bovine Brain

PrP^(Sc) is isolated from a fresh brain sample of a cow that exhibitedsymptoms of a neurological disorder consistent with the presence ofPrP^(Sc). Approximately 10 g of brain tissue is used to produce ahomogenate. The brain tissue is flash-frozen in liquid nitrogen, andthen homogenized using a standard mortar and pestle technique todissociate the tissue for further extraction procedures. Phosphatebuffered saline (PBS) pH 7.4 containing 4% (w/v) sodiumdodecylsarcosinate (sarcosyl), an ionic surfactant, is added to thebrain homogenate in a 1:5 (v/v) ratio to the brain homogenate. Asolution of 4% sodium phosphotungstic acid (PTA) and 170 mM MgCl₂, pH7.4, is added to the buffered homogenate solution to a finalconcentration of 0.2% PTA. The sample is exposed to the PTA for 16 hoursat 30° C. on a rocking platform. At the end of 16 hours, Proteinase K isadded to the solution providing a final concentration of 25 mg/ml, andthe sample is incubated for one additional hour at 37° C. The additionof proteinase K generally increases the efficience of the precipitationof PrP^(Sc), in part by degrading other remaining proteins includingPrP^(c).

Following incubation, the sample is transferred to 1.5 ml sterile tubes,with approximately 1 ml aliquots of the PTA-homogenate solution pertube. The sample is centrifuged at 10,000 g in a table top centrifuge(Eppendorf) for 40 minutes at room temperature. The supernatant isdecanted from the tubes, and each pellet is resuspended in sterile waterto the desired overall protein concentration. Protease inhibitors areadded to the solution: PMSF to a concentration 0.5 mM, Aprotinin to afinal concentration of 2 mg/ml, and Leupeptin to a final concentrationof 2 mg/ml. The protease inhibitors protect the sample from degradationunder certain storage conditions. An aliquot of protein for current useis stored at 4° C. The remaining protein is aliquoted, and stored at-20° C.

The total protein content of the pellet is reduced 100 fold as comparedto similar procedures known in the art. The PrP^(Sc) or PrP 27-30content of this pellet represents approximately 40-60% of the totalprotein. This procedure thus results in a protein sample highly enrichedin PrP^(Sc) protein species.

EXAMPLE 3 Isolation of PrP^(Sc) from Human Brain

PrP^(Sc) is isolated from brain samples of a deceased individualsuspected of being affected with a prion-based neurological disorder.Approximately 5 g of human brain tissue is used to produce a homogenate.The homogenate is produced using a dounce homogenizer to dissociated thetissue for protein extraction. A solution of 0.24 mM Triton-X, anon-ionic surfactant, in PBS pH 7.4 is added to a final 1:1 (v/v) ratio.A solution of 4% sodium phosphotungstic acid (PTA) and 170 mM MgCl₂, pH7.4, is added to the buffered homogenate solution to a finalconcentration of 0.3% PTA. The sample is exposed to the PTA for 8 hoursat 37° C. on a rocking platform.

Following incubation, the sample is transferred to 2.0 ml sterile tubes,with approximately 1 ml aliquots of the PTA-homogenate solution pertube. The sample is centrifuged at 14,000 g in a table top centrifuge(Brinkmann) for 30 minutes at room temperature. The supernatant isdecanted from the tubes, and each pellet is resuspended in a 25 mg/mlproteinase K solution. The tubes are incubated for one additional hour,rocking, at 37° C. The sample is again centrifuged at 14,000 g in atable top centrifuge for 30 minutes at room temperature. The supernatantis decanted, and the sample resuspended in 100 ml of sterilized water.Concentration can be determined using spectrophotometric techniques. ThePrP^(Sc) or PrP 27-30 content of this pellet represents approximately40-60% of the total protein.

EXAMPLE 4 Isolation of PrP^(Sc) from Sheep Brain

A brain sample from a sheep exhibiting neurological disorders isisolated and homogenized using a Polytron automated homogenizer.Approximately 1 gram of protein is homogenized in this fashion. PBS pH7.4 containing 1% (w/v) sodium dodecyl sulfate (SDS) is added to thebrain homogenate in a 5:1 (v/v) ratio. A solution of 4% sodiumphosphotungstic acid (PTA) and 170 mM MgCl₂, pH 7.4, is added to thebuffered homogenate solution to a final concentration of 0.25% PTA. Thesample is exposed to the PTA for 1 hour at 45° C. on a rocking platform.

Following incubation, the sample is transferred to 1.5 ml sterile tubes,with approximately 1 ml aliquots of the PTA-homogenate solution pertube. The sample is centrifuged at 20,000 g in a table top centrifuge(Eppendorf) for 20 minutes at room temperature. The supernatant isdecanted from the tubes, and each pellet is resuspended in sterile waterto the desired overall protein concentration. An aliquot of protein forcurrent use is stored at 4° C. The remaining protein is aliquoted, andstored at -20° C. The PrP^(Sc) or PrP 27-30 content of this pelletrepresents approximately 40-60% of the total protein.

EXAMPLE 5 Rendering infectious material non-infectious

Samples of scrapie-infected 5% (w/v) Syrian hamster brain homogenates,prepared in PBS, pH 7.4 (no Mg or Ca) and containing 2% (w/v) of sodiumdodecylsarcosinate (Sarcosyl), were mixed with stock solution containing4% sodium phosphotungstate (NaPTA) and 170 mM MgCl₂, pH 7.4. Samplescontaining final 0.2-0.3% (w/v) of PTA were incubated for 1-16 hrs at37° C. on a rocking platform.

Eppendorf tubes containing typically 1 ml samples were centrifuged at14,000 g in a table top centrifuge (Eppendorf) for 30 min at roomtemperature. The supernatant was decanted and pellet resuspended in H₂ Ocontaining protease inhibitors (PMSF 0.5 mM; Aprotinin and Leupeptin, 2μg/ml each). The starting brain homogenate, the homogenate containingPTA, the supernatant of PTA precipitated brain homogenate, andresuspended pellet were assayed for prion infectivity by incubation timeassay in Syrian hamsters. The results are presented as titer/ml in FIG.1.

Prion infected material is rendered non-infectious by usingphosphotungstic acid to precipitate infectious prions fromscrapie-infected Syrian hamster brain homogenates. Brain homogenate infinal concentration of 5%, containing 2% Sarcosyl, was prepared frombrains of scrapie-infected (isolate Sc237) Syrian hamsters (LVG/LAK),mixed with final 0.3% of phosphotungstic acid (5% BH/0.3% PTA), and spunfor 30 min at 14,000 g, and separated into pellet (Pell) and supernatant(Sup)--as shown in FIG. 1. PTA precipitated 99% of the infectious prionspresented in the material in the pellet. Columns and bars representingdata obtained from independent experiments are shown in FIG. 1.

The instant invention is shown and described herein what is consideredto be the most practical, and preferred embodiments. It is recognized,however, that departures may be made therefrom which are within thescope of the invention, and that obvious modifications will occur to oneskilled in the art upon reading this disclosure.

What is claimed is:
 1. A method of concentrating a constricted form of aprotein which occurs in a constricted conformation and a relaxedconformation, comprisingadding a complexing agent to a sample whereinthe complexing agent selectively complexes with the constrictedconformation of the protein; mixing the complexing agent with the samplefor a time sufficient to form complexes between the agent and aconstricted form of a protein; centrifuging the sample containing thecomplexes; and treating the sample with a protease which selectivelydegrades the relaxed conformation of the protein, wherein the treatingis carried out prior to or after centrifuging.
 2. The method of claim 1,wherein the constricted form of the protein is associated with apathogenic disorder; and further wherein the sample is from the braintissue of an animal suspected of having a central nervous systemdisorder; and still further wherein the mixing is carried out for a timeand in a manner which evenly distributes the complexing agent in thesample.
 3. The method of claim 1, wherein the centrifuging is carriedout at about 10,000 to 20,000 g for about 10 minutes or more, the sampleis homogenized brain tissue containing PrP^(Sc) and the complexing agentis phosphotungstic acid.
 4. The method of claim 1, furthercomprising:decanting away a supernatant obtained after centrifuging;obtaining a pellet resulting from the centrifuging and decanting;resuspending the pellet in water and protease inhibitor to obtain asuspension; and analyzing the suspension.
 5. The method of claim 4,wherein the analyzing is carried out using a process selected from thegroup consisting of: an SDS PAGE, a Western blot, a dot blot, andvisualization via electron microscopy.
 6. The method of claim 1, whereinthe protein is a PrP protein, and the protease is Proteinase K.
 7. Themethod of claim 6, wherein the treating is carried out prior to thecentrifuging.
 8. A method of concentrating PrP^(Sc) present in a sample,comprising:adding phosphotungstic acid or a salt thereof to a sample;mixing the phosphotungstic acid or salt thereof with the sample; andcentrifuging the sample containing the phosphotungstic acid or saltthereof to precipitate out and thereby concentrate PrP^(Sc) in thesample.
 9. The method of claim 8, wherein the phosphotungstic acid isadded in an amount so as to provide a concentration of about 0.05 to 10%of phosphotungstic acid, and wherein the mixing is carried out for atime and in a manner which evenly distributes the phosphotungstic acidin a sample.
 10. The method of claim 8, where the centrifuging iscarried out about 10,000 to 20,000 g for about 10 minutes or more. 11.The method of claim 8, further comprising:treating the sample withProteinase K.
 12. The method of claim 8, further comprising:decantingaway supernatant obtained after centrifuging; and obtaining a pelletresulting from the centrifuging and decanting.
 13. The method of claim12, further comprising:resuspending the pellet in water and proteaseinhibitor to obtain a suspension; and analyzing the suspension.
 14. Themethod of claim 13, wherein the analyzing is carried out using a processselected from the group consisting of an SDS PAGE, a Western blot, a dotblot, and visualization via electron microscopy.
 15. The method of claim8, wherein the sample is a sample of homogenized brain tissue from ananimal suspected of having PrP^(Sc) in its brain tissue.
 16. The methodof claim 8, wherein the sample is a pharmaceutical composition comprisedof a pharmaceutically active drug and pharmaceutically acceptablecarrier.
 17. A method of rendering a sample non-infectious wherein thesample comprises a protein which occurs in a first, infectious,constricted conformation and a second, non-infectious, relaxedconformation, comprising:adding a complexing agent to the sample whereinthe complexing agent selectively complexes with the constrictedconformation and is added in an amount such that complexing agentmolecules are available for all molecules of constricted conformation ofthe protein present in the sample; mixing the complexing agent with thesample for a time and in a manner which evenly distributes thecomplexing agent in the sample and allows complexes to be formed betweenmolecules of complexing agent and molecules of the constrictedconformation of the protein; centrifuging the sample for a time and at aspeed to precipitate complexes from a sample supernatant formed bycentrifuging; separating the precipitated complexes from the samplethereby rendering the sample non-infectious.
 18. The method of claim 17,wherein the protein is PrP and the constricted conformation is PrP^(Sc)and the relaxed conformation is PrP^(c).
 19. The method of claim 17,wherein the complexing agent is phosphotungstic acid or a salt thereof.20. The method of claim 17, further comprising:treating the sample witha protease which selectively degrades the relaxed form of the protein.